1. Field of the Invention
The present invention relates to the field of binding of molecules such as transcription factors to regions of nucleic acids, steroid hormone usage, steroid receptors and their corresponding response elements. Reagents are provided to allow methods involving direct detection of binding of a molecule, determining response element targeting by activated steroid receptors, screening for steroid agonists and antagonists, and monitoring levels of steroid agonists and antagonists in biological samples.
2. Background Art
Steroid receptors are hormone-dependent activators of gene expression. Steroid receptors mediate the action of steroid hormones (e.g., glucocorticoids, estrogens, progestins, testosterone, mineralocorticoids and 1,25-dihydroxycholecalciferol) in human tissues. After activation with the cognate ligand, receptors bind to chromatin in the nucleus and modulate the activity of target cellular genes. The binding of receptors to these target sequences is a key step in steroid function. Currently, this interaction can only be detected by indirect methods, such as reporter assays that detect the result of transcriptional activation coupled with transfection methods that introduce DNA sequences with receptor binding sites.
It is generally accepted that the unliganded glucocorticoid receptor (GR) resides in the cytoplasm, and that hormone activation leads both to nuclear accumulation and gene activation. (Gasc, J. -M. and Baulieu, E. E. (1987) in Steroid Hormone Receptors: Their Intracellular Localisation, ed. Clark, C. R. (Ellis Horwood Ltd., Chichester, England), pp. 233-250; Beato, M. (1989) Cell 56, 335-344; Carson-Jurica, M. A., Schrader, W. T. and O""Malley, B. W. (1990) Endocr. Rev. 11, 201-220; Gronemeyer, H. (1993) in Steroid Hormone Action, ed. Parker, M. G. (Oxford University Press, New York), pp. 94-117; Tsai, M. J. and O""Malley, B. W. (1994) Annu. Rev. Biochem. 63, 451-486; Akner, G., Wikstrom, A. C. and Gustafsson, J. A. (1995) J. Steroid Biochem. Mol. Biol. 52, 1-16), and references therein. However, the mechanisms involved in nuclear translocation and targeting of steroid receptors to regulatory sites in chromatin have been poorly understood. It has previously been difficult to discriminate between the ability of a given receptor mutant, or a given receptor/ligand combination, to participate in the separate processes of receptor activation, nuclear translocation, sequence-specific binding, and promoter activation.
Proteins have previously been labeled with fluorescent tags to detect their localization and sometimes their conformational changes both in vitro and in intact cells. Such labeling is essential both for immunofluorescence and for fluorescence analog cytochemistry, in which the biochemistry and trafficking of proteins are monitored after microinjection into living cells (Wang, Y. L. and Taylor, D. L., eds. (1989) Methods Cell Biol. 29). Traditionally, fluorescence labeling is done by purifying proteins and then covalently conjugating them to reactive derivatives of organic fluorophores. The stoichiometry and locations of dye attachment are often difficult to control, and careful repurification of the proteins is usually necessary. If the proteins are to be used inside living cells, a final challenging step is to get them across the plasma membrane via micropipet techniques or various methods of reversible permeabilization. Furthermore, in previous hormone studies broken cell preparations or antibody tags in fixed cell preparations were used, both techniques that cause enormous disruption of cell structures.
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a molecule whose natural function seems to be to convert the blue chemiluminescence of the Ca2+-sensitive photoprotein aequorin into green emission (Ward, W. W. (1979) in Photochemical and Photobiological Reviews, ed. Smith, K. C. (Plenum, New York), 4:1-57). GFP""s absorption bands in the blue (maximally at a wave length of 395 nm with weaker absorbance at 470 nm) and emission peak in the green (at 509 mn) do not arise from a distinct cofactor but rather from an internal p-hydroxybenzylideneimidazolidinone chromophore generated by cyclization and oxidation of a serine-tyrosine-glycine sequence at residues 56-67 (Cody, C. W., Prasher, D. C., Westler, W. M., Prendergast, F. G. and Ward, W. W. (1993) Biochemistry 32, 1212-1218). The gene for GFP was cloned (Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G. and Cormier, M. J. (1992) Gene 111, 229-233), and the encoded protein consists of 238 amino acid residues (molecular weight 27 kD). Heterologous expression of the gene has been done in Escherichia coli (Heim, R., Prasher, D. C. and Tsien, R. Y. (1994) Proc. Natl. Acad. Sci. U.S.A. 91,12501-12504); Inouye, S. and Tsuji, F. I. (1994) FEBS Lett. 341, 277-280), Caenorhabditis elegans (Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994) Science 263, 802-805), and Drosophila melanogaster (Yeh, E., Gustafson, K. and Boulianne, G. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 7035-7040; Tannahill, D., Bray, S. and Harris, W. A. (1995) Dev. Biol. 168, 694-697 and plants (Hu, W. and Cheng, C. L. (1995) FEBS Lett. 369, 331-334; Baulcombe, D. C., Chapman, S. and Santa Cruz, S. (1995) Plant J. 7, 1045-1053). Recently, chimeric genes encoding N- and C-terminal fusions of the Drosophila exuperantia (exu) gene product, Exu (Wang, S. and Hazelrigg, T. (1994) Nature 369, 400-403), actin Act88F gene (Barthmaier, P. and Fyrberg, E. (1995) Dev. Biol. 169, 770-774), and a nuclear localization signal (Davis, I., Girdham, C. H. and O""Farrell, P. H. (1995) Dev. Biol. 170, 726-729); of the yeast microtubule and spindle pole associated disl gene product (Nabeshima, K., Kurooka, H., Takeuchi, M., Kinoshita, K., Nakaseko, Y., and Yanagida, M. (1995) Genes Dev. 9, 1572-1585) and an RNA binding protein Np13p (Corbett, A. H., Koepp, D. M., Schlenstedt, G., Lee, M. S. Hopper, A. K. and Silver, P. A. (1995) J. Cell Biol. 130, 1017-1026); and of a mammalian ion channel protein, NMDAR1 (Marshall, J., Molloy, R., Moss, G. W., Howe, J. R. and Hughes, T. E. (1995) Neuron 14, 211-215), microtubule-associated protein, MAP4 (Olson, K. R., McIntosh, J. R. and Olmsted, J. B. (1995) J. Cell Biol. 130, 639-650), and a secretory protein, chromogranin B (Kaether, C. and Gerdes, H. H. (1995) FEBS Lett. 369, 267-271) have been constructed fused to GFP. However, none of these chimeric proteins have been to transcription factors or co-factors and no suggestions have been made as to the usefulness of such a fusion to study physiologically relevant interaction on an amplified DNA target. Furthermore, none of these reports indicated a successful use of GFP in mammalian cells.
Many human diseases result from aberrant steroid function, and many disease states, i.e., inflammation, are treated with glucocorticoid and other steroid derivatives. A large number of drugs have been developed whose function is based on the ability to interact with and activate steroid receptors. The identification and characterization of these compounds is a laborious, time-consuming and expensive process involving years of work. Even with a large investment of resources, the true behavior of these compounds in living cells is not understood.
The present invention allows observation for the first time of in vivo target sites within a higher eukaryotic nucleus for trans-regulatory molecules, such as transcription factors, e.g., glucocorticoid receptor (GR). The visualization of physiologically relevant in vivo target sites for any transcription factor to date has not previously been accomplished. The present invention provides a powerful method for identification of any single target site in a higher eukaryotic genome, comprising roughly 60,000-80,000 genes (Bird, A. P. (1995) Trends Genet. 11:94-100), using a singly fluorescently-labelled regulatory factor, which has not been considered previously. Discriminating direct versus indirect interaction between a regulatory molecule and its putative regulatory site is critical for the development of highly specific drugs directed against trans-regulatory factors. Traditionally, the methodology for showing potentially direct interactions involves nuclease or chemical protection experiments and transient co-transfection experiments of the putative regulator and its regulated site. While this approach indicates potential direct interaction, it does not necessarily imply direct interaction. Alternatively, the approach of making compensatory mutations between the regulatory sequences as well as the DNA binding specificity has been used in an attempt to demonstrate direct regulatory interaction (Schier, A. F. and Gehring, W. J. (1992) Nature 356:804-807), an extension of the principles of second site suppression in genetics to molecular biology. However, such an approach makes enormous assumptions of our understanding of sequence-specific recognition by sequence-specific DNA binding proteins in vivo, which certainly would not be valid for many systems, since many profound developmental events are governed by exquisite interactions to fine tune the system regarding, for example, concentration gradients of trans-regulatory factors The present invention allows a simple and straight-forward manner in which direct interaction between a sequence-specific DNA binding protein or its co-factor and its putative regulatory site in the in vivo genomic context can be addressed. With this simple inventive methodology, novel classes of drugs directed not only against members of the steroid-ligand-dependent transcription factors but to new classes of drugs that target other transcription factors or their co-factors can be screened.
Additionally, the present invention provides the first opportunity to observe and monitor gene targeting specifically of steroid receptors in living cells wherein binding of the steroid receptor to its response element target can be observed distinctly from translocation of steroid receptor. The invention therefore provides for many relevant analyses, such as real-time determination of steroid activity in subjects as well as screening of compounds for response element binding/targeting capabilities as distinct from translocation capabilities. Such methods have implications in many diseases associated with steroid hormones, such as endocrine disorders, rheumatic disorders, collagen diseases, dermatological diseases, allergic states, ophthalmic diseases, respiratory disease, hematologic disorders, neoplastic disease, edematous states, gastrointestinal diseases and neurological conditions, and in other uses such as prevention of rejection of transplanted tissues.
The present invention provides a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor.
The present invention further provides a chimeric protein comprising a fluorescent protein fused to a transcription factor. The present invention also provides a chimeric protein comprising a fluorescent protein fused to a steroid receptor.
The instant invention provides an isolated nucleic acid encoding a chimeric protein comprising a fluorescent protein fused to a transcription factor and an isolated nucleic acid encoding a chimeric protein comprising a fluorescent protein fused to a steroid receptor.
The instant invention also provides a cell containing a nucleic acid encoding a chimeric protein comprising a fluorescent protein fused to a transcription factor and a cell containing a nucleic acid encoding a chimeric protein comprising a fluorescent protein fused to a steroid receptor.
The instant invention provides a method of screening for a compound that binds to a selected nucleic acid comprising:
a. contacting compound fluorescently labeled by a fluorescent protein with a cell having a plurality of copies of the nucleic acid in an array such that the nucleic acid can be directly detected when bound by fluorescently labeled compound; and
b. directly detecting the location of fluorescence within the cell, fluorescence aggregated at the site of the nucleic acid array indicating a compound that binds to the selected nucleic acid.
The present invention also provides a method of characterizing a ligand""s effect on cellular localization of a compound to which the ligand binds in a cell comprising:
a. contacting the ligand with a cell having the compound fluorescently labeled by a fluorescent protein and
b. directly detecting the location of fluorescence within the cell, the location of fluorescence in the cell indicating the localization effect of the ligand on the compound.
Additionally provided is a method of determining a binding site for a DNA-binding protein comprising:
a. contacting the DNA-binding protein fluorescently labeled by a fluorescent protein with a cell having a plurality of copies of a nucleic acid having a putative binding site in an array such that the putative binding site can be directly visualized when bound by the fluorescently labeled DNA-binding protein, and
b. directly detecting the location of fluorescence within the cell, the presence of fluorescence aggregated at the putative binding site indicating a binding site to which the DNA-binding protein binds.
The present invention also provides a method of screening for a ligand that activates gene targeting of a steroid receptor in the nucleus of a mammalian cell comprising:
a. contacting the ligand with a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor and the cell further comprising a nucleic acid encoding a chimeric protein wherein a fluorescent protein is fused to the steroid receptor; and
b. directly detecting the location of fluorescence within the cell, fluorescence aggregated at the site of the steroid receptor response element array in the nucleus indicating a ligand that activates the gene targeting of a steroid receptor in the nucleus of a mammalian cell.
The present invention provides a method of screening for a ligand that activates the translocation of a steroid receptor to the nucleus in a mammalian cell comprising:
a. contacting the ligand with a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor and the cell further comprising a nucleic acid encoding a chimeric protein wherein a fluorescent protein is fused to the steroid receptor; and
b. directly detecting the location of fluorescence within the cell, the location of fluorescence aggregated in the nucleus indicating a ligand that activates the translocation of a steroid receptor to the nucleus in a mammalian cell.
The instant invention provides a method of detecting in a biological sample the presence of an agonist of a steroid receptor comprising:
a. contacting the sample with a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor and the cell further comprising a nucleic acid encoding a chimeric protein wherein a fluorescent protein is fused to the steroid receptor; and
b. directly detecting the location of fluorescence within the cell, the location of fluorescence aggregated at the site of the steroid receptor response element array in the nucleus indicating the presence of an agonist of the steroid receptor in the sample.
The present invention also provides a method of detecting in a biological sample the presence of an antagonist of a steroid receptor comprising:
a. contacting the sample and an agonist of the steroid receptor with a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor and the cell further comprising a nucleic acid encoding a chimeric protein wherein a fluorescent protein is fused to the steroid receptor; and
b. directly detecting the location of fluorescence within the cell, the absence of fluorescence substantially aggregated at the site of the steroid receptor response element array in the nucleus indicating the presence of an antagonist of the steroid receptor in the sample.
The present invention provides a method of monitoring the level of an agonist of a steroid receptor in a subject comprising:
a. periodically obtaining a biological sample from the subject,
b. contacting the sample with a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor and the cell further comprising a nucleic acid encoding a chimeric protein wherein a fluorescent protein is fused to the steroid receptor; and
c. directly detecting the location of fluorescence within the cell, a decrease in fluorescence aggregated at the site of the steroid receptor response element in the nucleus in a later-obtained sample relative to an earlier-obtained sample indicating a decrease in level of the steroid agonist of the steroid receptor in the sample and an increase in fluorescence aggregated at the site of the steroid receptor response element in the nucleus in a later-obtained sample relative to an earlier-obtained sample indicating an increase in level of the steroid agonist of the steroid receptor in the sample.
The instant invention provides a method of monitoring the balance between levels of an agonist of a steroid receptor and an antagonist of the steroid receptor in a subject comprising:
a. periodically obtaining a biological sample from the subject,
b. contacting the sample with a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor and the cell further comprising a nucleic acid encoding a chimeric protein wherein a fluorescent protein is fused to the steroid receptor; and
c. directly detecting the location of fluorescence within the cell, an increase in fluorescence aggregated at the site of the steroid receptor response element in the nucleus in a later-obtained sample relative to an earlier-obtained sample indicating an increase in level of the steroid agonist relative to level of the steroid antagonist in the sample, and a decrease in fluorescence aggregated at the site of the steroid receptor response element in the nucleus in a later-obtained sample relative to an earlier-obtained sample indicating an increase in level of the steroid antagonist of the steroid receptor relative to level of the steroid agonist in the sample.
The instant invention also provides a method of determining an effective dosage of a steroid receptor agonist in a subject comprising:
a. transferring into a set of cells from the patient a nucleic acid encoding a chimeric protein comprising a fluorescent protein fused to a steroid receptor;
b. contacting the cells in the set with one of a selected range of dosages of the steroid agonist; and
c. directly detecting location of fluorescence in the set of cells, a dosage capable of locating fluorescence substantially in the nucleus indicating an effective dosage of steroid receptor agonist.
The present invention provides a method of determining an effective dosage of a steroid receptor agonist to maintain steroid receptor activation for a selected period of time in a subject comprising:
a. administering to the subject a dosage of the steroid receptor agonist,
b. periodically obtaining a biological sample from the subject,
c. contacting the sample with a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor and the cell further comprising a nucleic acid encoding a chimeric protein wherein a fluorescent protein is fused to the steroid receptor; and
d. directly detecting the location of fluorescence within the cell, a dosage that maintains the location of fluorescence at the site of the steroid receptor response element array in the nucleus for the selected period of time indicating an effective dosage.
The present invention also provides a method of determining an effective dosage of a steroid receptor antagonist to abrogate agonist activity for a selected period of time in a subject comprising:
a. administering to the subject a dosage of the steroid receptor agonist,
b. periodically obtaining a biological sample from the subject;
c. contacting the sample with a mammalian cell having a plurality of steroid receptor response elements in an array such that the response element can be directly detected when bound by fluorescently labeled steroid receptor and the cell further comprising a nucleic acid encoding a chimeric protein wherein a fluorescent protein is fused to the steroid receptor; and
d. directly detecting the location of fluorescence within the cell, a dosage that prevents the location of fluorescence at the site of the steroid receptor response element array in the nucleus for the selected period of time indicating an effective dosage.
The present invention also provides a method of detecting a defect in a response pathway of a steroid receptor in a subject comprising transferring into a cell from the subject a nucleic acid functionally encoding a chimeric protein comprising a fluorescent protein fused to the steroid receptor and detecting the location of fluorescence within the cell as compared to the location of fluorescence within a normal, control cell transfected with the nucleic acid, a difference in location of fluorescence within the cell of the subject as compared to location of fluorescence within the normal, control cell indicating a defect in the response pathway of the steroid receptor.
The instant invention provides a method of determining whether a defect in a response pathway of a steroid receptor in a subject is in translocation of the steroid receptor to a cell nucleus, comprising transferring into a cell from the subject having the defect a nucleic acid functionally encoding a chimeric protein comprising a fluorescent protein fused to the steroid receptor and detecting the location of fluorescence within the cell, the location of fluorescence substantially in the cytoplasm of the cell indicating the defect is in translocation of the steroid receptor to the nucleus.